1. Field of the Invention
The present invention relates in general to the optical non-contact sensor field and, specifically, to a system and method for using an optical label independent detection (LID) biosensor (e.g., waveguide grating-based biosensor) to monitor in real time compound-induced mass redistribution in living cells, including agonist-induced G protein coupled receptor (GPCR) desensitization and translocation within living cells, as well as morphological changes of adherent cells. Particularly, the present invention relates to a system and method for using a LID biosensor to screen compounds against a GPCR within living cells.
2. Description of Related Art
Today an optical-based biosensor like a surface plasmon resonance (SPR) sensor or a waveguide grating-based sensor enables an optical label independent detection (LID) technique to be used to detect a biomolecular binding event at the biosensor's surface. In particular, the optical-based biosensor enables an optical LID technique to be used to measure changes in a refractive index/optical response of the biosensor which in turns enables a biomolecular binding event to be detected at the biosensor's surface. In fact, these optical-based biosensors along with different optical LID techniques have been used to study a variety of biomolecular binding events including oligonucleotides interactions, antibody-antigen interactions, hormone-receptor interactions, and enzyme-substrate interactions (for example).
In general, the optical-based biosensor includes two components: a highly specific recognition element and an optical transducer that converts a molecular recognition event into a quantifiable signal. The traditional studies performed with optical LID techniques have been associated with direct optical methods which include the use of: surface plasmon resonance (SPR) sensors; grating couplers; ellipsometry devices; evanescent wave devices; and reflectometry devices. For a detail discussion about each of these direct optical methods reference is made to the following documents:    Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements of Electrostatic Biopolymer Adsorption onto Chemically Modified Gold Surfaces,” Anal. Chem., 1997, 69:1449-1456.    Morhard et al., “Immobilization of antibodies in micropatterns for cell detection by optical diffraction,” Sensors and Actuators B, 2000, 70, 232-242.    Jin et al., “A biosensor concept based on imaging ellipsometry for visualization of biomolecular interactions,” Analytical Biochemistry, 1995, 232, 69-72.    Clerc and Lukosz “Direct immunosensing with an integrated-optical output grating coupler” Sensors and Actuators B 1997, 40, 53-58.    Brecht & Gauglitz, “Optical probes and transducers,” Biosensors and Bioelectronics, 1995, 10, 923-936.The contents of these documents are incorporated by reference herein.
To date, there have been relatively few reports describing the use of optical LID techniques to perform cell-based assays. For example, SPR biosensors have been used to investigate the adhesion and spreading of animal cells as described in the following document:    J. J. Ramsden, S. Y. Li, J. E. Prenosil and E. Heinzle, “Kinetics of adhension and spreading of animal cells” Biotechnol. Bioeng. 1994, 43, 939-945.
And, SPR biosensors have been used to investigate ligand-induced cell surface and intracellular reactions of living cells as described in the following document:    M. Hide, et al. “Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor”, Anal. Biochem. 2002, 302, 28-37.
However, to date there has been no report concerning the use of optical LID techniques to monitor compound-induced mass redistribution within adherent cells including agonist-induced translocation of G protein coupled receptors (GPCRs) within living cells. It would be desirable if this was possible, because GPCRs, a family of cell surface receptors, are the most common targets that new drug compounds are designed against. And, because GPCRs can transduce exogenous signals (i.e., the presence of stimuli such as a new drug) into intracellular response(s) which makes them extremely valuable in the testing of new drugs.
GPCRs participate in a wide array of cell signaling pathways. Ligand binding initiates a series of intracellular and cellular signaling events, including receptor conformational changes, receptor oligomerization, G protein activation (GDP-GTP exchanges on Gα subunit, Gα and Gβγ disassociation, G protein decoupling from the receptor, generation of Gα- and Gβγ-signaling complexes), and downstream signaling activation that leads to second messenger generation (Ca2+ mobilization, inositoltriphosphate generation, and/or intracellular cAMP level modulation) and ultimately results in changes of specific gene expression. Ligand-mediated GPCR activation also leads to the desensitization of GPCRs from the cell surface and trafficking of many intracellular proteins, as well as changes in phenotypes, morphology and physical properties of the target cells. These changes could be static, long-lasting or dynamic (e.g., cycling or oscillation). Distinct signaling events exhibit significantly different kinetics ranging from milliseconds (e.g., GPCR conformational changes) to tens of seconds (e.g., Ca2+ flux) to even tens of minutes (e.g., gene expression, or morphological changes). Current GPCR assays include ligand-receptor binding, second messenger (Ca2+, cAMP of IP3) assays, protein interaction assays, translocation assays and reporter gene assays. Since GPCR activation ultimately leads to protein trafficking and/or morphological changes, methods that can study the action of any compounds through the GPCRs on cell surface and the consequent events (e.g., trafficking and/or morphological changes) of the effected cells would be desired. This need and other needs are addressed by the present invention.